The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 20 assays. Highlights range from performing 7 online validation assays, including hERG assay, MSTI/electrophile assay, p53 assay with or without liver microsomes, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. Moreover, many chemicals may need metabolic activation to show inhibitory effects. In collaboration with the CFSAN/FDA, NTP and EPA, the Systems Toxicology team has validated AChE enzyme-based assays with/without microsomes in both color matric and fluorescent readouts against a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition, which suggest that these methods can be used to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity. The team has profiled environmental chemicals that modulate the TGF/SMAD signaling pathway. From the primary screening, a group of known and novel compounds that modulate the TGF/SMAD pathway has been identified and further tested in assays, including the SMAD3 phosphorylation assay. These TGF/SMAD-active compounds were also compared to other developmental pathways, including the retinol signaling pathway (RSP) and the sonic hedgehog (SHH) pathway. To test 38 chemicals with developmental toxicity potential, the team has used in vitro co-culture angiogenesis assay previously optimized in a 1536-well plate format and found that the results from this co-culture angiogenesis assay were highly correlated with the in silico predictions. The estrogen-related receptor alpha (ERR-alpha) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. In collaboration with NIEHS, we identified two main groups of ERR antagonists (9 antineoplastic agents and 13 pesticides). Each of these compounds were screened and profiled in 15 antagonist assays (AR-HEK293, AR-MDA, CAR-HepG2, ER-HEK293, ER-MCF7, ER-HEK293, ERR-HEK293, FXR-HEK293, TRE-GH3, PGC/ERR-HEK293, PPAR-HEK293, PR-HEK293, RAR-C3H10T1/2, ROR-CHO, ShhGli1-3T3), 2 agonist assays (Nrf2/ARE-HepG2 and p53-HCT-116), and the MMP-HepG2 assay to identify any similar pattern of activity. The compounds were then treated in HepaRG cells and mRNA expression inhibition of 5 ERR downstream target genes (COX8A, IDH3, PPAR, COX4I1, and cytochrome c) was measured. The manuscript describing this study has been published in Molecules. In addition, the team has identified a potentially novel cluster of CAR activators containing nitazoxanide and tenonitrozole. These CAR agonists have been tested in human primary hepatocytes for the mRNA and protein expression of CYP2B6 and CYP3A4, as well as a CAR translocation. Four compounds were confirmed as CAR activators. The manuscript describing this study has been published in Toxicological Sciences.